The present invention relates to a novel luciferase derived from Decapoda. More specifically, the invention relates to a secretional luciferase from Oplophorus gracilirostris, which is composed of 19 kDa and 35 kDa proteins. The invention also relates to polynucleotides encoding at least one of these proteins, a recombinant expression vector comprising at least one of these polynucleotides, a host cell transformed with said vector, and process for producing said photoprotein.
The genes encoding luciferases or photoproteins which have been reported are listed in Table 1.
These photoproteins and luciferases are an industrially important protein and have been utilized, for example, as a reporter enzyme. Various methods for detecting an analyte using luminescent reactions of these enzymes have been developed, and also some apparatuses to be used in these methods have been improved and widespread. Among these known photoproteins and luciferases, however, there is no enzyme applicable to extensive purposes. Consequently, one has to choose a proper enzyme for individual purpose.
Among the prior art luminescent substrates (often referred to as luciferin), those having the determined structures are only the substrates represented by formulas (1)-(8): 
The luminescent substrates include species specific and species non-specific substrates. The minimum unit in the enzymatic bioluminescent reaction consists of a luminescent enzyme (luciferase), a luminescent substrate (luciferin) and molecular oxygen. A luminescent reaction which requires other components such as a co-enzyme or a supplemental molecule is also reported.
Examples of the luciferase with luminescence in minimum unit include those derived from Renilla, Cypridina and Gonyaulax. The luciferins corresponding to these luciferases have very complicated structures as shown in the above formulas (4) and (5). The methods for synthesizing Cypridina and Gonyaulax luciferins are already known, but yield is remarkably low due to their complicated synthesizing process. Though the luciferins extracted from natural products are,also used, they are very expensive with little industrial utility.
On the contrary, Renilla luciferin known as coelenterazine and derivatives thereof are commercially available and inexpensive, because various methods for the production thereof have been established.
Among the photoproteins in Table 1, a secretional luciferase is only Cypridina luciferase. The structure of the gene is reported in Thompson, E. M., et al., Proc. Natl. Acad. Sci. USA, 86, 6567-6571 (1989) and the application of the gene is reported in Inouye, S., et al., Proc. Natl. Acad. Sci. USA, 89, 9584-9587 (1992).
In the construction of a bioassay system such as a drug screening system, the secretional luciferase has an industrial advantage in that the luminescence activity can be detected in living cells without cell disruption using the luciferase extracellularly secreted as a reporter. Generally, a secretional protein is not particularly difficult to produce if a suitable host-vector system is selected. Further, the purification of a recombinant protein from cultured media is easier in comparison with the purification from a cell extract. Thus, mass production of the secretional luciferase may advantageously hold down purification costs involved.
A particularly useful luciferase involves a luminescent system wherein the luminescent reaction occurs only among the minimal unit, i.e. a luciferase, a luciferin and molecular oxygen, the luciferin being coelenterazine or a derivative thereof which is readily available, and the luciferase itself being a secretional protein. A protein and a gene of such luciferase are advantageous not only scientifically but also industrially. However, isolation of gene encoding such luciferase and expression thereof in a living cell has not been reported yet.
A luciferase derived from a luminous shrimp belonging to Decapoda has been reported as a secretional luciferase, the luminescent substrate of which is coelenterazine. It is known that a luminous shrimp involves a secretional luciferase (enzyme) and that a blue luminescence is emitted by the reaction of the luciferase, a luminescent substrate luciferin and molecular oxygen.
The detailed classification of globally living luminous shrimps is disclosed in Herring, P. J., J. Mar. Biol. Ass. U.K., 56, 1029-1047 (1976). The only biochemical study of luciferase of luminous shrimp is reported by Shimomura et al., on a luciferase of the luminous shrimp Oplophorus gracilirostris living in the Suruga Bay, Sizuoka, Japan (Biochemistry, 17, 994-998 (1978)). This study report discloses a luciferase having a molecular weight of 130,000 which is composed of the tetramer of a polypeptide having a molecular weight of 31,000. The literature also reports that the luciferase has a quantum yield of 0.32 at 22xc2x0 C., a high specific activity of 1.75xc3x971015 Photons/s. mg, an optimum light emission at 40xc2x0 C. and an excellent heat stability. It also describes that the luminescent reaction proceeds in a wide range of pH.
The luciferin in the luminescent reaction of Oplophorus luciferase is coelenterazine represented by the above formula (2), which is also a luminescent substrate in the luminescent reactions of Renilla luciferase and a photoprotein, aequorin. The most important difference between these luminescent enzymes and Oplophorus luciferase is that Oplophorus luciferase has very broad substrate specificity in comparison with those of Renilla luciferase and aequorin. Oplophorus luciferase is more preferable than other luciferases, because it can utilize as a substrate bisdeoxycoelenterazine which is an analogue of coelenterazine and is available at a low cost.
However, either the protein structure or the gene structure of Oplophorus luciferase of a secretional type has not been elucidated. This is because living luminous shrimp, which are mostly living in the deep-sea, are very difficult to obtain in a large amount. Furthermore, population of the shrimp is decreasing due to the environmental changes. Therefore, construction of gene library from Oplophorus gracilirostris as well as early isolation of a gene encoding Oplophorus luciferase are desired.
An object of the present invention is to provide polynucleotides encoding Oplophorus luciferase and a luminescent subunit thereof.
Another object of the invention is to provide the recombinant secretional Oplophorus luciferase and the subunit encoded by the polynucleotides.
Another object of the invention is to provide an expression vector containing the polynucleotide(s) and a host transformed with the vector.
Further object of the invention is to provide a method for producing the recombinant Oplophorus luciferase or the recombinant photoprotein.
As a result of the isolation and purification of a secretional luciferase from Oplophorus gracilirostris, the present inventor found that Oplophorus luciferase is composed of 19 kDa and 35 kDa proteins. Then, the partial amino acid sequence of each protein was determined and the cloning of the proteins was carried out based on the information of their partial amino acid sequences. The genes coding for the two proteins was successfully cloned and the nucleotide sequences of the genes and the amino acid sequence of these proteins encoded by the gene could also be determined. Further, the present inventor has succeeded in preparing the expression vector containing the polynucleotide coding for each gene and a host such as a microorganism or a cultured animal cell which had been transformed with the vector. These proteins could be recombinantly produced by culturing the host cell or by in vitro translation system using the recombinant expression vector.